Kinetic tracking of Plasmodium falciparum antigen presentation reveals determinants of protein export and membrane insertion

Intracellular malaria parasites export many proteins into their host cell, inserting several into the erythrocyte plasma membrane to enable interactions with their external environment. While static techniques have identified some surface-exposed proteins, other candidates have eluded definitive localization and membrane topology determination. Moreover, both export kinetics and the mechanisms of membrane insertion remain largely unexplored. We introduce Reporter of Insertion and Surface Exposure (RISE), a method for continuous nondestructive tracking of antigen exposure on infected cells. RISE utilizes a small 11 aa NanoLuc fragment inserted into a target protein and detects surface exposure through high-affinity complementation. We tracked insertion of CLAG3, a malaria parasite protein linked to nutrient uptake, throughout the P. falciparum cycle in human erythrocytes. Our approach also revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved surface exposure by up to 50-fold, revealing that both size and charge of the extracellular epitope influence membrane insertion. Insertion of parasite proteins at the host cell surface and antigen accessibility is regulated by multiple factors, enabling intracellular parasite survival and immune evasion under a broad range of conditions.